Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development

Abstract

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/βcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.

Figure 1. Effects of inactivating Sp6 and Sp8 in limb development.

The external aspect (top row) and skeletal preparations of the forelimb (middle row) and hindlimb (bottom row) of newborns are shown for each genotype (genotypes indicated at the top). In the absence of the four functional alleles of Sp6 and Sp8 (A–C), or when only one functional allele of Sp6 remains (D–F) no limbs form. Underdeveloped hip bones with rudimentary ilium and ischium form when one functional allele of Sp6 is present (F). Animals with only one functional allele of Sp8 (G–I) display a split hand/foot malformation phenotype with occasional absence of the radius (H′) and more severe phenotype in the hindlimb (I). The digit tips in these limbs show conical nails (J), compare with normal digits (K). Abbreviations: s, scapula; h, humerus; r, radius; u, ulna, f, femur, t, tibia, fi, fibula, is: ischium il, ilium.

Figure 2. RT-qPCR quantification of Sp6 and Sp8 transcripts in the limb ectoderm of E10.5 control embryos.

Histogram bars represent the average expression values after normalization to the ubiquitously expressed 18s-RNA (standard deviation shown as error bars). Sp8 (red) exhibits a higher level of expression than Sp6 (blue) both in forelimbs (FL) and in hindlimbs (HL) and both factors are expressed at higher level in the forelimb than in the hindlimb.

Figure 3. Msx2Cre removal of Sp8 on a Sp6 deficient background.

The external aspect (top row) and skeletal preparations of the forelimb (middle row) and hindlimb (bottom row) of newborns are shown for each genotype (genotypes indicated at the top). Msx2Cre conditional removal allows transient expression of Sp8 in both forelimbs and hindlimbs which results in Sp8 conditional mutant (D–F) displaying a milder limb phenotype than ubiquitous mutants (A–C). One single conditional allele of Sp8 in the forelimb (G–H) seems to be equivalent to both functional alleles of Sp6 (A–B) while in the hindlimb is not sufficient for limb development (C, I). This conditional allele of Sp8 in addition to one single allele of Sp6 permits the formation of the three PD segments of the limb although with a single digit (J–L). Finally, one conditional allele of Sp8 plus a normal allele Sp8 results in SHFM (M–O).

Figure 4. Fgf8 is not detected in double Sp6;Sp8 mutants.

ISH to transverse sections through the level of the forelimbs at the stage indicated at the top and with the probe indicated on the left. Genotypes are also marked at the top of the figure. In the absence of Sp6 and Sp8, Fgf8 expression in the limb ectoderm is never detected as shown at E9.5 (A–B), E10.5 (I–J) and E11.5 (O–P). However, Bmp4 (C–D), Fgf10 (E–F) and Msx2 (G–H) are normally activated at E9.5 but not maintained at later stages (K–N). Note that the initial budding of the double mutant is similar to normal (A–H) but further growth is impaired (I–N) and complete regression has occurred by E11.5 (O–P). In all panels dorsal is up and distal to the right.

Figure 5. Effects of inactivating Sp6 and Sp8 genes on cell survival and AER morphogenesis.

(A–B) TUNEL assay showing abundant apoptotic cells (green) both in the mesoderm and ectoderm of the E10.5 double mutant forelimb bud (B) compared to control (A). (C–D) semithin longitudinal section of araldite embedded control and double mutant limb buds showing the thickening in the ventral ectoderm of mutants. The insert in D shows a lower magnification to appreciate the ventral position of the ectoderm thickening in mutants. (E–F) Confocal images of double immunohistochemistry for Laminin-b, marking the basement membrane (green) and E-cadherin expressed specifically in the ectoderm (red) showing that the cells accumulated in the ventral limb ectoderm of mutant embryos are of ectodermal origin. (G–H) Confocal images of Connexin 43 immunostaining showing the enrichment of gap junctions in the control AER (green dots) but not in the double mutant AER. All the panels show forelimb buds at E10.5. In the immunostainings, the nuclei are counter stained with DAPI (blue).

Figure 6. Effects of inactivating Sp6 and Sp8 genes on dorsal-ventral limb patterning.

ISH to transverse sections through the level of the forelimbs at the stage indicated at the top and with the probe indicated on the left. Genotypes are also marked at the top. Note that, contrary to controls (A, E), Wnt7a is not restricted to the dorsal ectoderm in double mutant embryos (B, F). Accordingly, En1 expression is undetectable in the ventral limb ectoderm of mutant embryos (D, H) compared to controls (C, G). The arrowheads and arrows mark the distal limit of Wnt7a and En1 expression, respectively.

Figure 7. Molecular and morphological analysis of Sp6−/−;Sp8+/− mutant limb buds.

(A–C) WMISH for Fgf8 and Bmp4 showing irregular activation in the limb bud ectoderm of Sp6^−/−;Sp8^+/− E10 (A), E10.5 (B) and E11.5 (C) forelimb buds compared to control littermates. Note the irregular early activation and predominant posterior maintenance of Fgf8 and Bmp4 expression, except for a residual focus of anterior expression (red arrowheads). (D, D′, D″) ISH for Wnt7a and En1 to consecutive (7 microns apart) sections of control and mutant E10.5 forelimb buds (D, D′ and D″). Note the variable expansion of Wnt7a into the ventral ectoderm always associated with a corresponding proximal restriction of En1 (D′, D″) indicated by red arrowheads (D′) and red arrows (D″). (E–E′) ISH for Lmx1b and Fgf8 in consecutive sections of control and Sp6−/−;Sp8+/− E11.5 forelimb buds. The Lmx1b expression invades the ventral mesoderm distally under the broad and flat AER. (F–G) Hematoxylin-Eosin stained transverse histological sections at the autopod and zeugopod level of E15.5 control (F) and Sp6−/−;Sp8+/− (G) limbs. Some of the individual muscles and tendons are labeled. Abbreviations: EC, extensor digitorium communis; FDS, flexor digitorium sublimis; FDP, Flexor digitorium Profundus; ECR, extensor carpi radiallis; m, metacarpal; R, radius; U, ulna.

Figure 8. Tp63 and Dlx5 expression in mutant limb buds.

(A–F) Tp63 and Dlx5 expression is normally detected in the limb ectoderm of control (A, B), Sp6−/−;Sp8+/− (C, D) and Sp6−/−;Sp8−/− (E, F) mutants although Dlx5 is downregulated. (G–H) Immunostaining for Tp63 (green) showing expression in the Sp6^−/−;Sp8^−/− double mutant limb bud similar to wild type littermate. All the panels show longitudinal sections of E10.5 forelimb buds.

Figure 9. Illustration showing the correlation between the Sp6/Sp8 gene dose and the severity of the limb phenotype.

Blue boxes represent the Sp6 alelles and red boxes the Sp8 alelles. Grey boxes represent null alleles and boxes with a red to grey graduation represent conditionally removal with the Msx2Cre allele.

Figure 10. Regulatory pathways mediated by Sp6 and Sp8.

Sp6 and Sp8 are necessary mediators of the Wnt/βcatenin-dependent induction of Fgf8 in the limb ectoderm. In addition, these two factors also collaborate with BMP signaling in the induction of En1 in the ventral limb ectoderm. Finally, Sp6 and Sp8 may also act downstream of Tp63 and Dlx genes.